The long-term goal of this research is to understand how repetitious elements, here tandem arrays in particular, are packaged into heterochromatin for silencing. The genomes of higher eukaryotes are made up primarily of repetitious DNA, including both dispersed transposable elements (TEs) and simple tandem repeats. Silencing of repetitious DNA is essential for maintaining genome integrity and regulated gene expression. However, sporadic local expansion of tandem repeats can occur, resulting in inappropriate gene silencing and consequent human genetic disability [e.g., Friedreich's ataxia (FRDA), Fragile X syndrome (FMR)]. A key objective of this proposal is to elucidate the mechanisms that lead to silencing of different types of repetitious sequences in a common genomic environment. This is being done using ?landing pad? sites in the Drosophila melanogaster (fruit fly) genome, assessing the ability of different repetitious DNA sequences to trigger heterochromatin formation, as shown by silencing (variegation) of an adjacent hsp70-driven white reporter gene. We will use genetic and biochemical approaches to determine the critical trans-acting factors for silencing three different types of repeats: a single copy TE remnant (1360); a 256-copy tandem array of a foreign sequence (the 36-bp lacO element); a 310-copy tandem array of GAA, modeling the FRDA mutation, and a planned >200-copy tandem array of CGG, modeling the FMR mutation. We anticipate that HP1a will play a role in heterochromatin formation in all cases, but that other components may differ; for example, different H3K9 histone methyltransferases and/or different histone deacetylases may be utilized. To elucidate the commonalities and differences among the silencing mechanisms, we will pursue three aims. 1. Identify trans-acting factors required to silence these tandem repeats: both a targeted and an unbiased forward genetic screen will be used to identify components of the heterochromatin system required for each case of repeat-induced silencing. ChIP-qPCR experiments and experiments tethering specific components (e.g. histone modifiers) to the repeat will be used to confirm the inferences from genetics. 2. Investigate the mechanism of targeting tandem repeats for silencing. How are repeats identified? While a piRNA mechanism is likely for TE 1360, an R-loop-based mechanism has been suggested for tandem repeats, and will be investigated. 3. Determine whether tandem repeat-induced heterochromatin formation can be reversed by drug treatment. We have shown that an inhibitor of histone deacetylases (nicotinamide) reduces the silencing triggered by the lacO repeats. The fly system should prove valuable both for screening and for checking the mechanisms by which ?epigenetic? drugs impact the silencing system. A deeper understanding of how tandem repeats are silenced will provide new insights into epigenetic regulation, and facilitate development of strategies to manipulate silencing for human health management.